Synergistic inhibition of the calcification of glutaraldehyde pretreated bovine pericardium in a rat subdermal model by FeCl3 and ethanehydroxydiphosphonate: pre-incubation and polymeric controlled release studies

Calcification is a frequent cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde-pretreated porcine aortic valves or glutaraldehyde-pretreated bovine pericardium (GPBP). We investigated the hypothesis that ferric chloride (FeCI,) and sodiumethanehydroxydiphosphonate (EHDP) may act synergistically to prevent bioprosthetic tissue calcification. Pre-incubations and controlled release systems were studied individually. FeC13EHDP polymeric controlled release matrices were formulated using silicone rubber and evaluated for in vitro release kinetics at pH 7.4 and 37°C. The effects of Fe-EHDP synergism on GPBP calcification were investigated with 21 d subdermal implants in 3 wk-old male rats. Results demonstrated that levels of Fe3+ and EHDP uptake, measured in GPBP tissues pre-incubated first in an FeCI, solution (10e5 M) followed by an EHDP solution (0.1 M), were higher than in the reverse order of incubation. In the first series of rat implants, GPBP was pre-incubated in either FeCI, or Na,EHDP solutions, or sequential pre-incubations of first FeCI, and then Na,EHDP solutions, or the reverse. The inhibition of calcification was greatest when FeC13 (first preincubation, 10e5 M) was combined with Na*EHDP (second pre-incubation, 0.1 M) (1.78 f 0.2pg of Ca*+/mg of dried tissue) compared with the other pre-incubation groups: EHDP (first preincubation) combined with FeCI, (second pre-incubation) (21.7 k 6.4) FeCI, solution alone at 10m5 M (27.9 + 10.7), Na2EHDP solution alone at 0.1 M (52.3 + 11.9) and the control group (72.3 + 10.2). In a second series of implants, G.PBP specimens were co-implanted with individual controlled release systems containing one of the following formulations (weight percentage in silicone rubber): 1% FeCI,, 20% CaEHDP, 20% protamine sulphate, 1% FeCI,-20% CaEHDP, and 1% FeCI,-20% protamine sulphate. The 1% FeC13-20% CaEHDP silicone-rubber matrices were the most effective for inhibiting GPBP mineralization (13.7 t 3.0,ag Ca*+/mg of dried tissue) compared with non-drug silicone co-implant controls (74.7 f 5.56 pg Ca*+/mg of dried tissue) and other polymeric treatment groups (32.3 + 2.3-80.0 Ifr 19.7). No adverse effects on bone or overall growth of any treatment protocols were noted. Thus, combinations of FeC13 and EHDP, using either pre-incubations or polymeric controlled release, were synergistic for inhibiting GPBP calcification.